Establishing a functional and innovative AWA neuron-specific knock-down of importin-α3 in Caenorhabditis elegans and assess its role in chemotaxis behavior

Importins, distinguished into α and ß according to their structure and function, are regarded as the gatekeepers of the neural genome. They, in fact, aid in the transport of proteins such as transcription factors from the cytoplasm into the nucleus. Importantly, they allow for the integration of external cues and signals with the genome, prompting transcription. Although the molecular mechanisms regulating the interaction between importins and their cargos are well-established, the role of these interactions at the organism level and their effects on long-term transcription still remain elusive. Moreover, there is a knowledge gap concerning the function importins play in mature neurons and their role in the nervous system. It is intriguing to consider whether importins have functional specialization or individual importins are specific to certain neuronal populations or developmental stages. It is thus appealing to further elucidate the role of importins in the nervous system. In this study, we aimed to investigate the role importin α3 (ima-3) plays in AWA chemosensory neurons in Caenorhabditis elegans, a small, free-living nematode with a transparent body, short life cycle, fully annotated genome and 302 neurons. Importantly, it shares about 60% of disease-associated genes with mammals. C. elegans thus represents an incredibly advantageous in vivo model to investigate the role of importins in neurons. Here, we established innovative approaches to study the role ima-3 exerts in mature sensory neurons by investigating its effects in AWA neurons, essential for odor sensation. We first systematically mapped worm odor preference in two-choice chemotaxis assays of AWA-sensed odors. We then compared their behavior to that of an AWA sensory neuron-defective worm strain to verify the sensitivity of our assays and to ensure that even small effects resulting from ima-3 loss can be detected. After establishing worm chemotaxis behavior, we implemented the neuron-specific knock-down method for ima-3 targeting AWA neurons. Therefore, the sense and antisense sequences of ima-3 were lifted from the C. elegans genome along with the sequence of the AWA-neuron specific odr-10 promoter. Subsequently, the odr-10 promoter and the ima-3 sequences were fused to obtain two separate amplicons of sense and antisense ima-3 sequences, driven by the odr-10 promoter. These molecules are to be injected into the worms’ distal gonad arm to obtain progeny in which ima-3 is knocked-down by RNAi in AWA neurons specifically, allowing us to study its role in these neurons. We also lifted and fused the GFP sequence with the odr-10 promoter and aim to co-inject this amplicon into the worms’ distal gonad arm to visualize and quantify changes to AWA neuronal morphology, acting as a selective marker. Significantly, together, the Podr-10::GFP fragment and the AWA neuron-specific ima-3 knock-down will allow us to assess the effects of ima-3 loss on neuronal morphology and our extremely sensitive behavioral assays will permit us to detect even small difference due to this loss. This study has laid the grounds to systematically map the function of importin α3 and other importins and their interactors in mature neurons. This will shed light onto the mechanisms governing the importin-cargo interaction and their effects both at the organism level and on long-term transcription, paving new ways to combat neurological disease.